Screening,Verification And Function Research The Proteins Of THP-1 Cells Interaction With Salmonella Typhimurium EF-Tu

Salmonella is a kind of the gram negative bacteria, posing a great ham to human and animal health, and the infection rate of S. typhimurium is at the first place in Salmonella,about 1.5 billion people infected each year(WHO), as a common source of contamination of food. According to the surveys, the enteritis was caused by Salmonella, but the main part of Salmonella is S. typhimurium, and the trend of infection, incidence was significantly increased.As the facultative intracellular bacterium, S. Typhimurium can grow, breed and migratein mononuclear-phagocyte system, avoiding contacted by antibodies, also the Salmonella containing vesicles provide a safe and stable environment, which will guarantee Salmonella persistent exist in the body. At present, studies have confirmed that EF-Tu protein has many biological functions and the ability will induce the host immune response in variety of bacteria species. Put the hypothesis: EF-Tu may be recognized and captured as an important immunogen in the process of S. Typhimurium infecting host cells, making a role between bacteria and host.In this study, S. Typhimurium EF-Tu protein as the bait, human mononuclear phagocytes cells(THP-1) as the host, using GST-pull down assy to screen and verify cell proteins interactions with EF-Tu, researching the relationship between the host and Salmonella, to clarify the pathogenic mechanism of Salmonella, to provide the theoretical foundation.Test is composed by the following parts:1. Prokaryotic recombination protein EF-TuPCR amplified tufA gene fragment following the S. Typhimurium genome, completing the construction of prokaryotic expression plasmid. The recombinant plasmid p GEX-6p-1-tufA/pET30a-tuf A was transferred into E. coli, induced expression and verified by SDSPAGE electrophoresis and Western blot. The results showed that the expressed proteins are consistent with the theoretical values.2. Preparation monoclonal antibodies of EF-TuProkaryotic protein His-EF-Tu was used to immunize BALB/c mice. The mouse antibody titer was detected whether it was up to the standard to preparation of of monoclonal antibody. 5 groups of hybridoma cells stably secreting monoclonal antibodies were obtained by cell fusion, indirect ELISA screening, five sub-clonal, and named respectively Tu1-A1、Tu1-C4、Tu1-H2、Tu4-C11 and Tu9-B9. The 5 groups of monoclonal antibody subtypes were Ig G1/?. The immunogenic protein His-EF-Tu, Salmonella typhimurium bacteria protein/ E. coli bacteria protein, recombinant protein GST-EF-Tu and Staphylococcus aureus protein(negative control) were analyzed by Western-blot, the relative molecular weight of the protein recognized after DAB colored were approximately the theory values respectively,also proved the specificity of EF-Tu monoclonal antibody.3. Location EF-Tu on S. typhimurium surfaceS. typhimurium was activated to logarithmic phase, takeing a little amount of bacteria liquid as sample. In order to localize EF-Tu, we use the indirect immunofluorescence assay,flow cytometric analysis and complement-neutralization test by EF-Tu MAb / Pc Ab. The results showed that EF-Tu existed on surface of S. typhimurium.4. Screening host cells proteins interaction with EF-TuThe fresh THP-1cells proteins and the purified EF-Tu protein were incubated, following to wash the product with PBS(PH7.4) for removing impurity proteins. The proteins complexes were eluted from GST Resin and analyzed by SDS-PAGE electrophoresis. After stained by coomassie brilliant blue and decolored by methanol, the result showed that two other proteins appeared in the gel. Sequencing the two proteins by mass spectrometry, pk M2 and e EF1α1were obtained.5. Verification interactionExtracting the THP-1 cells RNA to reverse transcription for c DNA, PCR amplified pk M2 and e EF1α1 gene fragments to construct the prokaryotic expression plasmid p ET28 apk M2 and p ET30a-e EF1α1, which were transformed into E. coli. After successfully expressed, a large number of prokaryotic recombinant proteins His-pk M2 and His-e EF1α1were purified using Ni column. Fresh EF-Tu protein was co-incubated with purified Hispk M2 and His-e EF1α1 respectively, also establishing the control group. After washing,remove the protein complexes from the Ni column to analyze by Western blot. Results showed that the experimental group appears a specific band while the control group is negative. At the same time, eukaryotic expression plasmid p Ds Red1-N1-tuf A expressing successfully, was detected by IFA in Hela cells. Establishing two transfection groups, Hela cells, containing eukaryotic expression plasmid, were stained by anti-pk M2 / FITC-Ig G antibodies and anti-e EF1α1 / FITC-Ig G antibodies severally, following observed at CLSM.The results showed that EF-Tu protein and pk M2 / e EF1α1 protein were co-localized in the cytoplasm, which may provide the feasibility for the interactions between two proteins at the space.6. To detect the change of expression of pkM2 and eEF1α1S. Typhimurium was inoculated into the cells plate, co-cultured with THP-1 cells at the ratio of 1:100, to infect the host. Took out the cells samples in different periods and detected the change of expression of pk M2 and e EF1α1 using q PCR, GADPH as the reference. Results showed that with the time of S. Typhimurium infecting cells increasing, the expression of pk M2 and e EF1α1 decreased.